EP0992243B1 - Fas-Antagonist FÜR DIE PROPHYLAXE ODER THERAPIE VON GVHD - Google Patents

Fas-Antagonist FÜR DIE PROPHYLAXE ODER THERAPIE VON GVHD Download PDF

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EP0992243B1
EP0992243B1 EP97909714A EP97909714A EP0992243B1 EP 0992243 B1 EP0992243 B1 EP 0992243B1 EP 97909714 A EP97909714 A EP 97909714A EP 97909714 A EP97909714 A EP 97909714A EP 0992243 B1 EP0992243 B1 EP 0992243B1
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fas
antibody
mouse
fas ligand
gvhd
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EP0992243A4 (de
EP0992243A1 (de
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Shigekazu Nagata
Takehiro Mochida Pharmaceutical Co. Ltd. YATOMI
Takashi Suda
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Osaka Bioscience Institute
Mochida Pharmaceutical Co Ltd
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Osaka Bioscience Institute
Mochida Pharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the extracellular domain of the human Fas ligand is highly homologous with the extracellular domain of rat Fas ligand (Takashi Suda et al., Cell, vol. 75, pages 1169 - 1178, 1993) and mouse Fas ligand (Tomohiro Takahashi et al., Cell, vol. 76, pages 969-976, 1994).
  • the human Fas ligand recognizes not only the human Fas but also the mouse Fas to induce the apoptosis, and vice versa, the rat Fas ligand and the mouse Fas ligand also recognize the human Fas to induce the apoptosis.
  • apoptosis Relationship between the apoptosis, in particular, the Fas-mediated apoptosis with various diseases and physiological phenomena has been recently indicated. For example, possibility has been indicated for involvement of abnormal Fas-mediated apoptosis in decline of T cell count in the patients suffering from AIDS, in the death of hepatocytes in viral fulminant hepatitis, in some types of autoimmune diseases, and the like.
  • Fas/Fas ligand system Involvement of the Fas/Fas ligand system is in functions other than the apoptosis has also been indicated. For example, possibility has been indicated for the Fas/Fas ligand system to react with neutrophils to develop proinflammatory action (Kayagaki, N. et al., Rinshou Meneki (Clinical Immunology), vol. 28, pages 667 - 675, 1996).
  • SIRS systemic inflammatory response syndrome
  • Ischemic reperfusion injury is found in practically all tissues and organs, and is involved in various diseases. Ischemic reperfusion injury is also a problem in preservation and transplantation of organs. Among such ischemic reperfusion injuries, those associated with infarction of liver, heart or kidney and those associated with surgery or transplantation, and in particular, tissue injury (such as cell necrosis) and dysfunction (such as cardiac arrhythmia) in the particular organ may lead to the death of the individual when they are serious, and therefore, such cases are a serious social problem. Various organ failures and ischemic reperfusion injuries of from the early stage to the late stage are known to be associated with production and secretion of IL-8.
  • endotoxin induces production of various cytokines in the body resulting in, for example, endotoxin shock in endotoxemia and sepsis as well as various organ damages including the liver damage (Dinarello, C.A. et al., J. American Medical Association, vol. 269, page 1829, 1993), and serious conditions are more than often induced. Observation of the apoptosis in such process and possibility of some involvement in such process of the Fas/FasL have been reported in experimental studies. However, it is not yet found how the Fas/FasL is involved in such failures.
  • NO nitrogen monoxide
  • IL-1 induces not only the synthesis of nitrogen monoxide (NO) of vascular smooth muscle cells but also apoptosis
  • apoptosis by IL-1 is inhibited by an inhibitor of the NO synthesis
  • Fas expression is induced by NO (Fukuo, K. et al., Hypertension, 27, 823 - 826, 1996).
  • apoptosis of cardiomyocytes is found in canine heart failure model and such apoptosis is associated with an increased Fas expression (Lab.
  • GVHD graft versus host disease
  • GVH reaction graft versus host reaction
  • Exemplary GVHDs are GVHD after bone marrow transplantation such as the incompatible bone marrow transplantation or the bone marrow transplantation in congenital immune deficiency syndrome; GVHD after organ transplantation; GVHD after blood transfusion, in which large amount of blood is transfused to a patient of hypoimmunity; and the like.
  • Fas-mediated apoptosis With regard to the involvement of the Fas-mediated apoptosis, there has been reported that no difference was found in survival time between the cases when the donor was spleen lymphocytes from a control mouse with normal Fas ligand and the cases when the donor was spleen lymphocytes from a gld mouse which is a Fas ligand-mutated mouse, and practically no damage in skin and liver was induced (Matthew, B., Barker, B. et al., J. Exp. Med., vol. 183, 2645 - 2659, 1996).
  • the report as described above utilizes spleen lymphocytes from a gld mouse for the material, and it is likely that the GVHD reaction is influenced by alteration in the amount of the expression of the factors other than the Fas ligand (such as perforin and TNF) as a substitute for the lack of the Fas ligand, and the results obtained may not necessarily reflect the genuine effect of the lack of the Fas ligand. Therefore, it is yet to be found out how the Fas-mediated apoptosis is involved in GVHD, and whether the substance which specifically inhibits the Fas-mediated apoptosis can be used as a therapeutic agent for the GVHD.
  • the Fas-mediated apoptosis is involved in GVHD, and whether the substance which specifically inhibits the Fas-mediated apoptosis can be used as a therapeutic agent for the GVHD.
  • the nonspecific immunosuppressives such as cyclosporin that have been used as a prophylactic or therapeutic agent of the GVHD generates nonspecific immunosuppression, and therefore, suffer from adverse side effects such as infections.
  • No therapeutic agent and no therapy for the GVHD wherein the GVHD is treated by inhibiting the Fas-mediated apoptosis are known to date.
  • no therapeutic agent and no therapy for the GVHD wherein the GVHD is treated by utilizing selective immunosuppression are known to date.
  • drugs mainly aims at thrombolysis and improvement of circulation, and no drug is available that directly prevents or treats the damage.
  • endotoxemia and sepsis steroid and proteolytic enzyme inhibitor, for example, are used in the case of shock, and no drug is currently available that directly prevents or treats the organ damage.
  • the drugs used for the diseases based on organ damage mainly aims at palliative treatment, and no drug is available that prevents or radically treats the diseases based on organ damage.
  • no prophylactic or therapeutic agent which are widely effective for various tissues and organs are available.
  • the inventors of the present invention have conducted an intensive study on function of the Fas/Fas ligand system and role of the apoptosis mediated by the Fas/Fas ligand system in various diseases, and found that conditions may be improved in various disease models by suppressing the actions of the Fas/Fas ligand system, and in particular, by suppressing the Fas/Fas ligand system-mediated apoptosis; and that, for example, that death of cardiomyocytes upon reperfusion after ischemia, onset of the GVHD associated with allogenic bone marrow transplantation, and organ damages caused by endotoxin are suppressed by an antagonist which inhibits the Fas-mediated apoptosis.
  • the present invention has been completed on the bases of such finding.
  • the agent of the present invention is used for the prophylaxis or therapy of GVHD.
  • the Fas antagonist is preferably at least one member selected from an anti-Fas ligand antibody, an anti-Fas antibody and a Fas derivative, and more particularly, the anti-Fas ligand antibody is preferably a humanized anti-Fas ligand antibody.
  • the present invention provides novel utilities of the Fas antagonist.
  • a Fas antagonist is a substance which has a suppressive or inhibitory action, and more particularly, a substance which suppresses or inhibits the biological actions of the Fas/Fas ligand system, and in particular the Fas-mediated cell apoptosis.
  • the present invention provides the use of a Fas antagonist which provides activity to interact with the extracellular domain of Fas ligand or the extracellular domain of the Fas and provides activity to inhibit the Fas-mediated apoptosis for the preparation of a pharmaceutical composition for the prophylaxis or therapy of graft versus host disease (GVHD).
  • a Fas antagonist which provides activity to interact with the extracellular domain of Fas ligand or the extracellular domain of the Fas and provides activity to inhibit the Fas-mediated apoptosis for the preparation of a pharmaceutical composition for the prophylaxis or therapy of graft versus host disease (GVHD).
  • the biological actions of the Fas/Fas ligand system, and in particular, the Fas mediated apoptosis relate to or contribute for the onset, remaining, or worsening of the symptoms or pathology of such a disease.
  • GVHD include GVHD after bone marrow transplantation such as incompatible bone marrow transplantation and bone marrow transplantation in congenital immune deficiency; GVHD after organ transplantation; posttransfusional GVHD such as GVHD after blood transfusion of a large amount to a patient of hypoimmunity.
  • GVHD is associated with organ or tissue failure based on GVH reaction, and diarrhea, exhaustion such as weight loss and thinning, exanthem, and liver dysfunction are observed.
  • GVHD is also associated with histological symptoms such as disorganization of bone marrow and lymphoid tissue and atrophy of intestinal villi.
  • the present invention is adapted for use in human, but also in animals other than human.
  • the Fas antagonist used in the present invention may be correctly referred to as an antagonist for the Fas/Fas ligand system, and the Fas antagonist is not limited to any particular type so long as it prevents or blocks the signal generation or transduction by the Fas at some stage, and it suppresses or inhibits the function or the biological action of the Fas/Fas ligand system, and in particular, the Fas-mediated apoptosis, and particularly, the Fas-mediated apoptosis by the Fas ligand.
  • the Fas antagonist may act through various mechanisms, and exemplary Fas antagonists are those inhibiting the action or the function of the Fas ligand or the Fas; those interacting with the extracellular domain of the Fas ligand or the extracellular domain of the Fas; those inhibiting the interaction between the Fas ligand and the Fas; those affecting the interaction between the intracellular domain of the Fas and an intracellular factor which interacts therewith; those suppressing the activity of the intracytoplasmic factor (such as ICE-like protease) which is involved in the signal transduction of the Fas-mediated apoptosis.
  • the Fas antagonists include both high molecular weight proteinaceous substance and low molecular weight compound.
  • exemplary Fas antagonists used in the present invention include a Fas derivative, an anti-Fas ligand antibody, an anti-Fas antibody, an antisense oligonucleotide for the mRNA or the gene of the Fas or the Fas ligand, a substance which interacts with the intracellular domain of the Fas, an ICE inhibitor, which are provided with the activity to inhibit the action of the Fas/FasL system, and in particular, the Fas-mediated apoptosis.
  • the Fas antagonist used in the present invention may be a Fas derivative, an anti-Fas antibody, or an anti-Fas ligand antibody which exhibits inhibitory action for the Fas-mediated apoptosis.
  • the Fas and the Fas ligand are preferably of human origin
  • the anti-Fas antibody and the anti-Fas ligand antibody are preferably a human anti-Fas antibody and a human anti-Fas ligand antibody, respectively
  • the anti-Fas ligand antibody is preferably a humanized anti-Fas ligand antibody.
  • the antibody used in the present invention may be either a polyclonal antibody or a monoclonal antibody, and the molecular species used in the present invention is not particularly limited.
  • the antibody used in the present invention may be either an antibody molecule of normal form or a fragment thereof which is capable of binding to the antigen to inhibit the Fas antigen-mediated apoptosis, for example, Fab, F(ab') 2 , Fv, or single chain Fv (scFv) which is the Fv of heavy chain linked to the Fv of light chain by an adequate linker to form a single chain.
  • the antibody used in the present invention may be an immunoglobulin of any class, subclass or isotype.
  • the antibody used in the present invention is not limited to any particular type as long as is capable of binding to the Fas ligand or the Fas antigen to inhibit the biological actions of the Fas/Fas ligand system, and in particular, the Fas antigen-mediated apoptosis.
  • the anti-Fas ligand antibody used in the present invention may be an antibody of any type (either monoclonal or polyclonal) and any origin produced by any appropriate process.
  • the anti-Fas ligand antibody is preferably a monoclonal antibody derived from a mammal.
  • the monoclonal antibody used in the present invention may be produced in any animal species so long as it is a mammal which may be human or non-human.
  • the monoclonal antibody from a mammal other than human may be the one from rabbit or other rodents.
  • the non-limiting preferable examples of such rodents are mouse, rat and hamster, and use of such animals facilitates a convenient production of the monoclonal antibody.
  • the monoclonal antibody may be the one which is capable of recognizing the antigen in a conventional immunoprocess such as radioimmunoassay (RIA), enzyme immunoassay (EIA, ELISA), immunofluorescent analysis, or the like, and whose activity of suppressing the apoptosis of the Fas antigen-expressing cell is measurable by an appropriate assay procedure described in International Patent Application Publication No. WO 95/13293.
  • RIA radioimmunoassay
  • EIA enzyme immunoassay
  • ELISA enzyme immunoassay
  • immunofluorescent analysis or the like
  • an example of the most preferable anti-Fas ligand antibody is mouse F919-9-18 antibody produced by hybridoma F919-9-18 which was originally deposited on June 22, 1995 in National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) (Accession No. P-15002) and transferred from the original deposition to the international deposition on May 9, 1996 (Accession No. FERM BP-5535).
  • the sequences of the variable regions of the antibody are shown in FIG. 1 (cDNA is described in SEQ. ID No. 1) and FIG. 2 (cDNA is described in SEQ. ID No. 2).
  • a monoclonal antibody When a monoclonal antibody is used in the present invention, such monoclonal antibody may be prepared by the process known in the art, for example, by using Fas antigen, Fas ligand, or a partial peptide thereof for the immunization antigen, immunizing an animal with such antigen in accordance with a conventional process, fusing the resulting immunized cell with a known parent cell by a conventional cell fusion process, and screening for the monoclonal antibody-producing cell by a conventional screening process.
  • Fas antigen Fas ligand
  • a partial peptide thereof for the immunization antigen
  • the mammal which is immunized with the immunization antigen is not limited to any particular type, and the mammal may be selected by considering the compatibility with the parent cell used in the cell fusion.
  • Exemplary animals are mouse, rat, hamster, and rabbit.
  • the immunization of the animal with the immunization antigen may be carried out by a known procedure. After the immunization and confirmation of the increase of the serum level of the desired antibody, the immunocytes are isolated from the animal, and subjected to cell fusion. The preferable immunocytes are splenocytes.
  • the parent cell to be fused with the immunocyte is not limited to any particular type. However, use of known mammal myeloma cell lines, and in particular, a mouse myeloma cell line such as P3-X63-Ag8-U1 (P3-U1) is preferred.
  • the cell fusion of the above-described immunocyte and the myeloma cell may be carried out basically in accordance with a known process such as the procedure of Milstein et al. (Milstein et al., Methods Enzymol. 73: 3 - 46, 1981).
  • the hybridoma is then screened for the one producing the target antibody used in the present invention and subsequently cloned.
  • the monoclonal antibody is obtained from the thus prepared hybridoma producing the monoclonal antibody used in the present invention by such procedures as cultivating the hybridoma according to the conventional method and obtaining the monoclonal antibody from the supernatant; or transplanting the hybridoma to a mammal compatible with the hybridoma for propagation, and obtaining the monoclonal antibody from the ascite of the mammal.
  • the former procedure is adapted for producing the monoclonal antibody of high purity
  • the latter procedure is adapted for producing the monoclonal antibody in a large amount.
  • the monoclonal antibody produced by such process for use in the present invention may be further purified by a known purification means such as salt precipitation, gel filtration, affinity chromatography.
  • the monoclonal antibody used in the present invention is not limited to the one produced by using a hybridoma, and may be the one produced by an antibody-producing cell immortalized by EBV or the one produced by a genetic engineering procedure.
  • the anti-Fas ligand antibody or the anti-Fas antibody used in the present invention is preferably a chimeric antibody or a humanized antibody which is an antibody intentionally altered for the purpose of reducing heteroantigenicity to human.
  • non-human monoclonal antibody such as mouse antibody is associated with defects when it is repeatedly used in treating a human.
  • the first defect is that the mouse monoclonal antibody has a relatively short circulation halflife and when used for human, the mouse monoclonal antibody will not develop other important functional properties of the immunoglobulin.
  • An exemplary such chimeric antibody is a chimeric antibody comprising the variable region from the monoclonal antibody of a mammal other than human such as mouse, and the constant region from the human antibody.
  • Such chimeric antibody may be produced by a known chimeric antibody production process, and in particular, by a genetic engineering process.
  • the Fas derivative used in the present invention is not limited to any particular type as long as it is capable of binding at least with the Fas ligand, or capable of inhibiting the Fas ligand-mediated apoptosis.
  • the Fas derivative may also be the one which comprises an amino acid sequence of a known Fas that has been arbitrarily mutated by substitution, deletion, addition or/and insertion, and which inhibits the biological actions of the Fas/Fas ligand system, and in particular, the Fas-mediated apoptosis, with the binding activity to the Fas ligand retained.
  • Exemplary Fas derivatives are the extracellular domain of a known Fas; a Fas antigen from which the transmembrane domain has been deleted; a chimeric protein of the extracellular domain of a Fas and another protein such as hFas-Fc which is a chimeric protein of the extracellular domain of human Fas and Fc fragment of human immunoglobulin.
  • the Fas derivative may be the one prepared by any production process by utilizing known Fas sequences and known gene engineering techniques. For example, the process for producing the human Fas-Fc is described in the Examples of International Patent Application Publication No. WO 95/13293.
  • Another preferable Fas derivative is the Fas having a deletion in its N terminal.
  • a Fas derivative coded in plasmids included in the E. coli which were originally deposited on March 14, 1996 in National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) (Accession Nos. P-15514 and P-15515) and transferred from the original deposition to the international deposition on March 6, 1997 (Accession No. FERM BP-5854 and Accession No. FERM BP-5855) (The accession Nos.
  • CCRC 940171 and CCRC 940170 are a derivative including the extracellular domain of the known human Fas from which N terminal sequence of from 1st to 29th amino acid has been deleted, and this highly active derivative is a preferable example of the effective component for the prophylactic/therapeutic agent of the present invention
  • partial nucleotide sequences in the vector (pM1304) including the nucleotide sequence of the cDNA coding for shFas(nd29)-Fc are described in FIGS. 5 to 9 and SEQ No.
  • the prophylactic/therapeutic agent used in the present invention may be in the form of a pharmaceutical composition or kit such as an injection or an oral medicine wherein the Fas antagonist is combined with at least one pharmaceutical carrier or medium such as sterilized water, physiological saline, a vegetable oil, a mineral oil, a higher alcohol, a higher fatty acid, or a nontoxic organic solvent; and optional additives such as an excipient, a colorant, an emulsifier, a suspending agent, a surfactant, a solubilizer, a nonadsorptive, a stabilizer, a preservative, a humectant, an antioxidative, a buffer, an isotonizing agent, or a pain relieving agent.
  • a pharmaceutical carrier or medium such as sterilized water, physiological saline, a vegetable oil, a mineral oil, a higher alcohol, a higher fatty acid, or a nontoxic organic solvent
  • optional additives such as an excipient, a colorant
  • the drug used in the present invention is parenterally administered either systemically or locally, and rapidly or gradually, for example, by intravenous, intramuscular, intraperitoneal, or subcutaneous injection.
  • the prophylactic/therapeutic agent used in the present invention should be administered at an adequate dose determined by taking the conditions and age of the patient as well as the administration route into the consideration. For example, an adequate divided dose may be selected in the range of approximately 0.1 to 100 mg/kg in the case of systemic administration.
  • the prophylactic/therapeutic agent used in the present invention is not limited to the administration route and the dose as described above.
  • the prophylactic/therapeutic agent used in the present invention may also contain a combination of two or more Fas antagonists, and may be used in combination with another drug.
  • the prophylactic/therapeutic agent used in the present invention may be formulated into a pharmaceutical preparation in a normal process.
  • an injection may be prepared by dissolving the purified Fas antagonist in a medium such as physiological saline or a buffer and optionally supplementing the solution with an additive such as an anti-adsorptive.
  • the preparation may also be in the form of a lyophilizate which is to be reconstituted before the use, and may contain any of the excipients that are generally used for facilitating the lyophilization.
  • the Fas antagonist used in the prophylactic/therapeutic agent of the present invention suppresses injury of the organ or tissue of the host and exhibits the effect of increasing the survival time and the survival rate in the heart ischemic reperfusion model as described in the Examples or in the GVHD model and in particular, in the GVHD model as described in the Examples.
  • the Fas antagonist also exhibits the suppressive effect for the increase of serum creatinine in the renal disease model, and the suppressive effect for the increase of GOT, GPT and the like which are indices of liver damage as well as the effect of increasing the survival rate in endotoxin-induced liver damage model.
  • the prophylactic/therapeutic agent used in the present invention administered to a patient suffering from such diseases will exhibit the effect of suppressing the injury and cell death, and in particular, the apoptosis of the cells of the particular organ or the tissue, and hence, the effect of preventing or treating the disease, the effect of alleviating the conditions and pathology associated with the disease, and the effect of suppressing the progress or worsening of the conditions.
  • the animals used in the Examples using the anti-Fas ligand antibody are rodents (mouse and rat), and therefore, the prophylactic and therapeutic effects are mainly demonstrated by using anti-mouse Fas ligand antibody.
  • the effects similar to those of the Examples are expected for the anti-human Fas ligand antibody and the humanized anti-human Fas ligand antibody when they are administered to human.
  • the agent used in the present invention is capable of preventing and treating GVHD and the conditions and pathology associated with the GVHD.
  • the GVHDs include GVHD after bone marrow transplantation such as incompatible bone marrow transplantation and bone marrow transplantation in congenital immune deficiency; GVHD after organ transplantation; posttransfusional GVHD such as GVHD after blood transfusion of a large amount to a patient of hypoimmunity; and the like.
  • the GVHD is associated with organ or tissue failure based on the GVH reaction, and diarrhea, exhaustion such as weight loss and thinning, exanthem, and liver dysfunction are observed.
  • the GVHD is histologically characterized by such symptoms as disorganization of bone marrow and lymphoid tissue and atrophy of intestinal villi.
  • the agent used in the present invention can also be used for prevention and treatment of such conditions and pathology associated with the GVHD.
  • the concentrate was dialyzed against 0.9% NaCl to obtain the purified shFas(nd29)-Fc.
  • hFas-Fc was purified in a similar manner.
  • the amount of the protein in each specimen was measured by Lowry's method using bovine serum albumin for the standard.
  • the elution fraction of the main peak was lyophilized, and dissolved in 70% formic acid for use as a sample, and the sample was analyzed for its N terminal amino acid sequence by Model 477A Protein Sequencing System - 120A PTH Analyzer (manufactured by Perkin Elmer Inc.).
  • PTH amino acid was detected by measuring absorbance at 270 nm in the ultraviolet region, and comparing the data by using the retention time of the standard PTH amino acid (manufactured by Perkin Elmer Inc.) which had been preliminarily separated by the same procedure. It was then found that the sample has the N terminal sequence of the human Fas antigen from which 29 N terminal amino acid residues had been deleted (Thr Gln Asn Leu Glu Gly Leu His His Asp).
  • CPK creatine kinase
  • the proportion (%) of the necrotic region to the ischemic region in the hFas-Fc treated group was lower than that of the control group (FIG. 13).
  • the creatine kinase (CPK) activity value of the hFas-Fc treated group was lower than that of the control group (FIG. 14).
  • hFas-Fc Effects of the hFas-Fc administration on the survival time was evaluated by administering 1, 3 or 10 mg/kg of hFas-Fc from the tail vein from the next day of the transplantation of the splenic lymphocytes from the donor mouse.
  • the control group was administered with the physiological saline supplemented with 0.1% human serum albumin (manufactured by Research Institute of Chemotherapy and Serotherapy (Foundation)), which was the diluent used for the hFas-Fc.
  • n was 5 for each group.
  • the survival time elongating effect was observed for the group treated with 10 mg/kg of hFas-Fc in comparison with the control group (FIG. 15). This group also exhibited less weight loss.
  • plasmid pM1304 16 ⁇ g of plasmid pM1304 was dissolved in 5.5 ⁇ l of 10mM Tris-HCl (pH 7.4)/1mM ethylenediaminetetraacetic acid solution.
  • F-12 Nutrient Mixture (Ham) medium was added to this solution (manufactured by GIBCO BRL Inc., hereinafter abbreviated as HamF12 medium) to a total volume of 800 ⁇ l to prepare solution A.
  • Ham F12 medium was added to 96 ⁇ l of Lipofect AMINE reagent (manufactured by GIBCO BRL Inc.) to a total volume of 800 ⁇ l to prepare solution B.
  • the DHFR positive cells expressing shFas(nd29)-Fc were then cloned in accordance with the method of Nobuhara et al. (Jikken Igaku (Experimental Medicine), vol. 5, No. 11, 1987, pages 1108 - 1112). More illustratively, about 100 single colonies were cloned by using penicillin cup, and subcloned on 48 well plates (manufactured by NUNC). The cells were incubated by substituting the medium at every 3 or 4 days with new MEM ⁇ (-) supplemented with 10% inactivated and dialyzed bovine fetal serum, and the cultivation was continued at a greater scale when the well became confluent.
  • the CHO(pM1304)72 cells were inoculated at 10 3 , 10 4 , and 10 5 cells/10 ml MEM ⁇ (-) medium supplemented with 10% inactivated bovine fetal serum/petri dish of 10 cm diam., and the medium was substituted with MEM ⁇ (-) supplemented with 10% inactivated and dialyzed bovine fetal serum containing 5nM MTX (manufactured by Lederle) to initiate the gene amplification.
  • the medium was substituted at every 3 or 4 days with new MEM ⁇ (-) supplemented with 10% inactivated and dialyzed bovine fetus serum containing 5nM MTX, and colonies of MTX resistant cells started to form in about 2 weeks.
  • the procedure adapted in producing the CHO(pM1304)72 cells was repeated to produce a cell line of high shFas(nd29)-Fc expression which is resistant to 5 nM of MTX (CHO(pM1304)72-105). Similar procedure was also repeated to produce a cell line of high shFas(nd29)-Fc expression which is resistant to 50 nM of MTX (CHO(pM1304)72-105-55).
  • Example 1(2) The procedure of Example 1(2) was repeated for purification of shFas(nd29)-Fc from the culture supernatant as described above by using protein A chromatography.
  • shFas(nd29)-Fc male BDF1 mice of 6 week old (Charles River Japan K.K.) were administered with shFas(nd29)-Fc at a dose of 10 or 30 mg/kg once in every two days for 12 days, namely, for 7 times in total from the tail vein to examine the effects.
  • the experiment was conducted by using three groups, namely, the control group, the group administered with 10 mg/kg of shFas(nd29)-Fc, and the group administered with 30 mg/kg of shFas(nd29)-Fc, and n was 3 for each group.
  • the control group was administered with 30 mg/kg of human serum albumin; the group of 10 mg/kg shFas(nd29)-Fc administration was administered with 10 mg/kg of shFas(nd29)-Fc and 20 mg/kg of human serum albumin; and the group of 30 mg/kg shFas(nd29) -Fc administration was administered with 30 mg/kg of shFas(nd29) -Fc.
  • the body weight was measured once in every two days from the day of the start of the administration. Blood was collected from orbital vein on 14th day from the start of the administration, and after counting blood cells, plasma was prepared to measure GOT, GPT and creatinine.
  • mice Male, 9 week old, Japan SLC K. K.
  • mice were used for the test animal by forming 3 groups of animals each comprising 5 mice.
  • the mice were administered from their tail vein with 0.2 ml of physiological saline having dissolved therein heat-killed Propionibacterium acness (P. acness) (RIBI IMMUNOCHEM RESEARCH, INC.) to a concentration of 5.0 mg/ml.
  • P. acness heat-killed Propionibacterium acness
  • mice 8 days after the administration, the mice were administered from their tail vein with shFas(nd29) -Fc prepared in Example 1 which had been diluted with a diluent (physiological saline containing 0.1% human serum albumin) at a dose of 0.3 mg/8 ml/kg or 1 mg/8 ml/kg.
  • the control group was administered with the diluent.
  • the mice were intraperitoneally administered with 0.2 ml of a solution of lipopolysaccharide (manufactured by Sigma) adjusted to a concentration of 5 ⁇ g/ml with physiological saline. 75 ⁇ l of blood was collected from orbital at 8 and 24 hours after the administration of the lipopolysaccharide.
  • the blood collected was mixed with 8.3 ⁇ l of 3.8% aqueous solution of sodium citrate, and centrifuged at 3000 rpm for 10 minutes. After the centrifugation, the resulting plasma was frozen by liquid nitrogen and stored at -30°C until the use.
  • GOT and GPT were measured with GOT-FA TestWako (manufactured by Wako Pure Chemical Industries, Ltd.), GPT-FA TestWako (manufactured by Wako Pure Chemical Industries, Ltd.), and an autoanalizer (Roche, COBAS FARA). It was then found that the GOT and GPT values in the group administered with 1 mg/8 ml/kg of shFas(nd29)-Fc were lower than the values of the control group to demonstrate the liver damage inhibitory effect.
  • mice Male, 5 week old BALB/c mice (Japan SLC K.K.) were used. The mice were administered from their tail vein with 0.2 ml of physiological saline containing 5.0 mg/ml of heat-killed Propionibacterium acness (P. acness) (RIBI IMMUNOCHEM RESEARCH, INC.). 10 days after the administration, the mice were administered from their tail vein with 0.2 ml of 50 ⁇ g/ml dilution of the extracellular domain of the human Fas ligand in physiological saline supplemented with 0.1% human serum albumin to prepare liver damage model mice.
  • P. acness heat-killed Propionibacterium acness
  • the extracellular domain of the human Fas ligand was obtained by transforming Pichia yeast (Pichia pastoris) GS 115 strain (manufactured by Invitrogen) with an expression plasmid which is a plasmid for Pichia yeast pPIC9 (manufactured by Invitrogen) having the DNA coding for the extracellular domain of the human Fas ligand incorporated therein; obtaining the culture supernatant of the transformant; and purifying the culture supernatant by means of salt precipitation with 80% saturation ammonium sulfate, protein A-Cellulofine affinity column having hFas-Fc bound thereto, Mono S (manufactured by Pharmacia) column, or other purification means (Tanaka, M. et al., Nature Medicine, vol. 2, pages 317 - 322, 1996).
  • the one prepared by the procedure described in Example 18 of International Patent Application Publication No. WO 95/13293 is similarly usable.
  • Mouse anti-human Fas ligand monoclonal antibody F919-9-18 produced by the hybridoma F919-9-18 with the Accession No. of FERM BP-5535 as described above was used.
  • the group administered with the mouse anti-human Fas ligand monoclonal antibody F919-9-18 was administered from the tail vein with 0.4 mg/kg or 1.2 mg/kg of F919-9-18 at 5 minutes before the administration of the extracellular domain of the human Fas ligand.
  • the control group was administered with the physiological saline supplemented with 0.1% human serum albumin which was the diluent for the anti-human Fas ligand antibody. n was 5 for each group.
  • a plasmid containing human elongation factor (EF) promoter, and in its downstream, the gene coding for the chimeric protein prepared by fusing the extracellular domain of mouse Fas ligand from soluble mouse Fas ligand WX2 (J. Immunology, vol. 157, pages 3918 - 3924, 1996) and the intracellular domain, the transmembrane domain, and a part of the extracellular domain (from N terminal to 78th amino acid) of mouse CD40 ligand was prepared (Mizushima-Nagata, Nucleic Acids Research, vol. 18, page 5322, 1990).
  • EF elongation factor
  • the plasmid was transfected in WR19L cell to obtain a recombinant cell W40LFL expressing the mouse Fas ligand on its cell membrane for use as the antigen to be administered.
  • Armenian hamsters were used for the animals to be immunized.
  • the Armenian hamsters were subcutaneously administered with 1 x 10 7 W40LFL mixed with Freund complete adjuvant, and a month later, subcutaneously administered with 2 x 10 7 W40LFL suspended in PBS, and in another a month later, administered with 5 x 10 6 W40LFL suspended in PBS from the foot pad.
  • lymph node cells were collected and fused with mouse myeloma cell P3-X63-Ag8-U1 (P3-U1). After selecting the hybridoma by HAT medium (hypoxanthine-aminopterin-thymidine), hybridomas FLIM4(#4-2), FLIM23 (#23-2), FLIM58(#58-11) whose supernatants had neutralizing activity for cytotoxicity of mouse Fas ligand were obtained from the survived hybridomas.
  • HAT medium hypoxanthine-aminopterin-thymidine
  • Hybridoma (#58-11) was cultured in serum-free medium Hybridoma-SFM (GIBCO BRL), and the culture supernatant was purified by protein A column (PROSEP-A, Bioprocessing) to obtain purified antibody FLIM58 (#58-11). Concentration of the protein was calculated from absorbance at 280 nm.
  • Example 11 Neutralizing activity of the anti-mouse Fas ligand antibody #58-11 for the mouse and rat Fas ligands
  • Neutralizing activity for anti-mouse Fas ligand antibody #58-11 was evaluated by an assay using release of 51 Cr for the index by repeating the procedure described in Example of International Patent Application Publication No. WO 95/13293.
  • Splenocytes from male, 7 week old ICR mice (Charles River Japan K.K.) and male, 11 week-old Wistar rats (Charles River Japan K.K.) were prepared by repeating the procedure of Example 4.
  • the resulting splenocytes were adjusted to a concentration of 2 x 10 6 cells/ml, and incubated overnight at 37°C in PRMI 1640 medium (GIBCO BRL) supplemented with 40 U/ml of recombinant human IL-2 (Boehringer Mannheim) and 10% inactivated FBS (JRH Bioscience) in the presence of 5% CO 2 gas.
  • the preparation of the target cells was conducted by a procedure similar to the procedure described in International Patent Application Publication No. WO 95/13293. More illustratively, a mouse Fas-expressing W4 cell was used for the target cell, and 10 6 W4 cells were incubated in PRMI 1640 medium supplemented with 20 ⁇ Ci of 51 Cr sodium chromate (NEN) at 37°C in the presence of 5% CO 2 gas for 2 hours for 51 Cr labeling.
  • PRMI 1640 medium supplemented with 20 ⁇ Ci of 51 Cr sodium chromate (NEN) at 37°C in the presence of 5% CO 2 gas for 2 hours for 51 Cr labeling.
  • Anti-mouse Fas ligand antibody #58-11 of various concentration were added to 1 x 10 6 activated mouse and rat splenocytes as described above, respectively, and the cells were incubated at 37°C for 30 minutes in the presence of 5% CO 2 gas. To the cultures were then added 1 x 10 4 W4 cells labeled with 51 Cr as described above, and after the centrifugation at 800 rpm for 2 minutes, the cells were incubated at 37°C for 4 hours in the presence of 5% CO 2 gas. The supernatants were measured for the amount of 51 Cr after the centrifugation at 1200 rpm for 5 minutes to evaluate the effect of the anti-mouse Fas ligand antibody #58-11.
  • shFas(nd29)-Fc which is the Fas derivative described in International Patent Application No. PCT/JP97/10502 was added in the same manner as the case of the anti-mouse Fas ligand antibody #58-11 as a positive control of the assay.
  • the thus obtained results were analyzed by a procedure similar to the assay procedure described in International Patent Application Publication No. WO 95/13293 using release of 51 Cr for the index.
  • the shFas(nd29)-Fc used as the positive control inhibits the cytotoxic activity of the activated mouse and rat splenocytes in a dose dependent manner at a concentration in the range of 0.3 ⁇ g/ml to 3 ⁇ g/ml, and that the activated mouse and rat splenocytes used exhibit FasL dependent cytotoxicity.
  • the anti-mouse Fas ligand antibody #58-11 inhibits the cytotoxic activity of the activated mouse and rat splenocytes in a dose dependent manner at a concentration in the range of 0.3 ⁇ g/ml to 3 ⁇ g/ml as in the case of the shFas(nd29)-Fc.
  • the anti-mouse Fas ligand antibody #58-11 inhibited the mouse and the rat Fas ligands.
  • mice Male, 8 week old DBA/1J mice and C3H/He mice (Charles River Japan K.K.) were used. The mice were administered from their tail vein with the anti-mouse Fas ligand antibody #58-11 at a dose of 100 mg/30 ml/kg. The control group was administered from the tail vein with physiological saline (Otsuka Pharmaceutical Co., Ltd.) at a dose of 30 ml/kg. n was 3 for both groups. Observation period was 7 days, and body weight measurement, hematological tests (red blood cell, white blood cell, platelet), and hematobiological tests (GOT, GPT, urea nitrogen), and autopsy with naked eye were conducted.
  • Rat heart ischemic reperfusion model was produced by repeating the procedure of Example 2.
  • Anti-mouse Fas ligand antibody #58-11 was diluted with physiological saline supplemented with 0.1% albumin, and administered to the rats from their right femoral vein at a dose of 1 mg/5 ml/kg immediately after the start of the reperfusion.
  • the control group was administered with IgG antibody (Cappel) derived from normal hamster serum at a dose of 1 mg/5 ml/kg.
  • the group administered with the anti-mouse Fas ligand antibody #58-11 consisted of 3 rats, and the control group consisted of 3 rats.
  • the measurement of the non-ischemic, ischemic and necrotic regions of the sections and the plasma creatinine kinase (CPK) measurement were conducted by repeating the procedure of Example 2.
  • the proportion (%) of the necrotic region in the ischemic region of the group administered with the anti-mouse Fas ligand antibody #58-11 was less than that of the control group (FIG. 17).
  • the plasma CPK value of the group administered with the anti-mouse Fas ligand antibody #58-11 was less than that of the control group (FIG. 18).
  • mice Male, 6 week old DBA/2 mice (Charles River Japan K.K.) were used for the host mice.
  • the host mice were intraperitoneally administered with 350 mg/kg of cyclophosphamide ((Shionogi & Co., Ltd., Endoxan) to induce myelosuppression.
  • cyclophosphamide (Shionogi & Co., Ltd., Endoxan) to induce myelosuppression.
  • a male B10.D2 mouse of 7 to 8 week old (Nihon SLC K.K.) was used for the donor mouse.
  • Spleen from the donor mouse was disintegrated in Hank's solution (manufacture by Nissui Seiyaku K.K.) with forceps and centrifuged, and the thus obtained cells were suspended in 0.017M Tris-0.747% ammonium chloride solution for selective hemolysis of erythrocytes.
  • the residual cells washed with Hank's solution were used as splenic lymphocytes from the donor mouse, and 3 x 10 7 cells/mouse were transplanted to the host mice from their tail vein on the next day of the cyclophosphamide administration as described above.
  • the survival rate at 8th day after the transplantation of the splenocytes were 63% in the GVHD negative group, 63% in the group administered with the anti-mouse Fas ligand antibody #58-11, and 38% in the group administered with the control antibody. Comparison of the survival rate with the group administered with the control antibody revealed the survival rate-improving effect in GVHD for the group administered with the anti-mouse Fas ligand antibody #58-11 (FIG. 19).
  • ICR mice Male, 6 week old ICR mice (Nihon SLC K.K.) were fixed in supine position on an operation plate after anesthetizing with pentobarbital, and abdominally dissected. Right kidney was extirpated from the abdominal opening, and ischemic condition was created by blocking the left renal artery and vein with a clip (VASCULAR CLIP AS-1, Kyowa Tokei Kogyo). After 30 minutes, the clip was undone and the blood was allowed to flow. Blood was collected from abdominal large vein 24 hours after the reperfusion, and measured for plasma creatinine and urea nitrogen.
  • VASCULAR CLIP AS-1 Kyowa Tokei Kogyo
  • Creatinine was measured by using Detamina CRE 55s (Kyowa Medics), and urea nitrogen was measured by using Urea Nitrogen B TestWako (Wako Pure Chemical Industries, Ltd.) in an autoanalyzer (COBAS FARA, Roche)
  • Anti-mouse Fas ligand antibody #58-11 was diluted with physiological saline supplemented with 0.1% BSA (SIGMA), and the mice were intravenously administered with the dilution at a dose of 0.3 mg/10 ml/kg or 3 mg/10 ml/kg immediately before the ischemia of the left renal artery and immediately after the reperfusion.
  • the control group was administered with the physiological saline supplemented with 0.1% BSA.
  • the physiological saline supplemented with 0.1% BSA was administered without creating the ischemic condition after the extirpation of the right kidney. All groups consisted of 8 cases.
  • the plasma creatinine value and the urea nitrogen value of the group administered with 3 mg/kg of the anti-mouse Fas ligand antibody #58-11 were less than those of the control group to demonstrate the inhibitory effect for the renal ischemic reperfusion injury (FIGS. 20 and 21).
  • the results as described above reveals that the prophylactic/therapeutic agent used in the present invention has excellent effects on GVHD.
  • the prophylactic/therapeutic agent used in the present invention exhibited no marked toxicity, and no toxicity was found at least in relation to the inhibition of biological actions of the Fas/Fas ligand system.
  • the prophylactic/therapeutic agent of the present invention containing a Fas antagonist as its effective component is useful in treating GVHD wherein the biological action of the Fas/Fas ligand system, and in particular, the Fas-mediated cell death, and particularly, the apoptosis is involved so long as the signal generation or transduction by Fas is blocked and the biological action of the Fas/Fas ligand system is suppressed.
  • the prophylactic/therapeutic agent used in the present invention containing a Fas antagonist as its effective component suppresses the biological action of the Fas/Fas ligand system, and in particular, the Fas-mediated cell death, and particularly, the apoptosis, and exhibits prophylactic or therapeutic effects for GVHD.
  • the prophylactic/therapeutic agent used in the present invention has no marked toxicity, and no toxicity has been found at least in relation to the inhibition of biological actions of the Fas/Fas ligand system. Therefore, the Fas antagonist used in the present invention should constitute a prophylactic/therapeutic agent for GVHD wherein the cell death, and in particular, apoptosis is involved.

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Claims (4)

  1. Verwendung eines Fas-Antagonisten, der eine Aktivität zur Verfügung stellt, um mit der extrazellulären Domäne des Fas-Liganden oder der extrazellulären Domäne des Fas zu wechselwirken, und eine Aktivität zur Verfügung stellt, um die Fas-vermittelte Apoptose zu hemmen, zur Herstellung einer pharmazeutischen Zusammensetzung für die Prophylaxe oder Therapie der Transplantation-versus-Wirtserkrankung (GVHD).
  2. Die Verwendung gemäß Anspruch 1, worin besagter Fas-Antagonist ein Fas-Derivat, ein Anti-Fas-Ligandenantikörper oder ein Anti-Fas-Antikörper ist.
  3. Die Verwendung gemäß Anspruch 2, worin besagter Anti-Fas-Ligandenantikörper ein humanisierter Anti-Fas-Ligandenantikörper ist.
  4. Verwendung eines Fas-Derivats, eines Anti-Fas-Ligandenantikörpers oder eines Anti-Fas-Antikörpers, das oder der eine Aktivität zur Verfügung stellt, um mit der extrazellulären Domäne des Fas-Liganden oder der extrazellulären Domäne des Fas zu wechselwirken, und eine Aktivität zur Verfügung stellt, um die Fas-vermittelte Apoptose zu hemmen, zur Herstellung einer pharmazeutischen Zusammensetzung für die Prophylaxe oder Therapie der Transplantationversus-Wirtserkrankung (GVHD).
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CN104662039B (zh) * 2012-07-18 2019-10-08 阿珀吉尼科斯股份公司 缩短的CD95-Fc变体
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